The broad, long-term goal of this work is to establish a comprehensive understanding of the role of pH in cardiac function at the cellular level. Changes in intracellular pH (pHi) and extracellular pH have profound effects on electrical activity, excitation-contraction coupling and contraction in the heart, which accounts in part for the arrhythmias and contractile dysfunction elicited by myocardial ischemia. Compared to the information available for ventricular muscle, much less is known concerning pHi regulation, the effects of pH on Ca 2+ handling, and intra- and intercellular H+ diffusion in cardiac myocytes that lack transverse tubules (Purkinje and atrial). The protocols described in this project focus primarily on these two cell types and are designed to help fill this gap using fluorescence imaging and patch pipette techniques. The specific aims include: 1. Characterize the effect of intracellular acidosis on excitation-contraction coupling in atria/and Purkinje myocytes. The goal here is to determine the effects of acidosis on Ca 2+ current (L and T type), the spatiotemporal profile of the Ca2+ transient, Ca 2+ handling by the sarcoplasmic reticulum and intracellular Ca 2+ diffusion in these cell types. The response of [Ca2+]i to spatially confined changes in pHi will also be studied. 2. Characterize the electrogenic properties of Na-HCO3 cotransport in ventricular, atrial and Purkinje myocytes. The objective here is to test the hypothesis that Na-HCO3 cotransport is electrogenic in these cell types. 3.Characterize the properties of pH_ regulatory systems in cardiac Purkinje myocytes. The objective here is to determine the kinetic properties and pH-dependence of Na-H exchange (NHE), Na-HCO3 cotransport (NBC) and HCO3-CI exchange (AE) in isolated Purkinje myocytes and to test the hypothesis that CI-OH exchange (CHE) is operational in this cell type. 4. Characterize intra- and intercellular H+ diffusion in atrial and Purkinje cells. The goal here is to determine the intracellular H+ diffusion coefficient within single cells and the junctional permeability H+ coefficient between pairs of electrically coupled myocytes and to test the hypothesis that both parameters are modulated by carbonic anhydrase.